title: In vitro assessment of anticryptosporidial efficacy and cytotoxicity of adenosine analogues using a SYBR Green real-time PCR method.
abstract: OBJECTIVE
  The aims of this study were to provide a cost-effective and valuable method for evaluating drug efficacy against Cryptosporidium parvum using a quantitative SYBR Green real-time PCR (qPCR) and to assess the efficacy of adenosine analogues as drug templates.
  
  
  METHODS
  C. parvum HNJ-1 strain growing in human ileocaecal adenocarcinoma cells was employed as an in vitro culture system. To normalize the DNA extraction efficiency, a specific plasmid was added to each sample before DNA purification; the genomic DNA of infected cells was quantified by qPCR using specific primers to confirm drug efficacy and cytotoxicity. To determine the mechanism of action, enzymatic inhibition analyses were conducted using C. parvum S-adenosyl-l-homocysteine hydrolase (CpSAHH) recombinant protein.
  
  
  RESULTS
  The dose-dependent growth inhibition of C. parvum was confirmed; 50% effective concentrations of neplanocin A (NPA) and 2-fluoroadenosine (2FA) were 139 μM and 0.842 μM, respectively. Cytotoxicity evaluation showed that the 50% growth inhibition concentration of 2FA was 1.18 μM; NPA did not exhibit any cytotoxicity up to 200 μM. The screening system revealed the specific but marginal efficacy of NPA and showed 2FA to be cytotoxic. Recombinant CpSAHH inhibition analyses showed that NPA competitively inhibited CpSAHH activity (K(i )= 0.395 μM), whereas 2FA did not.
  
  
  CONCLUSIONS
  This novel qPCR system confirmed not only drug efficacy against C. parvum but also cytotoxicity to host cells. Moreover, since the SYBR Green method is cost effective, it could therefore be used in a wide variety of clinical and research-oriented applications of Cryptosporidium analysis.
authors.affiliation: Department of Parasitology, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8640, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Arai
authors.forename: Isao
authors.initials: I
authors.lastname: Kimata
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakamoto
authors.forename: Masaharu
authors.initials: M
authors.lastname: Tokoro
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000633:toxicity; D000981:Antiprotozoal Agents / Q000494:pharmacology* / Q000633:toxicity; D052160:Benzothiazoles; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D016785:Cryptosporidium parvum / Q000187:drug effects* / Q000254:growth & development / Q000472:pathogenicity; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000379:methods / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011804:Quinolines; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000981:Antiprotozoal Agents; D052160:Benzothiazoles; D016054:DNA, Protozoan; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D000241:Adenosine